Cyp2j2 antagonists in the treatment of pain

ABSTRACT

The present invention pertains to novel treatments of neuropathic pain; in particular chemotherapy induced peripheral neuropathic pain (CIPNP). The invention provides antagonists cytochrome P450 epoxygenases (CYP), and more specifically antagonists of CYP2J2, as therapeutics for use in the treatment of neuropathic pain such as CIPNP. CYP2J2 antagonists were identified to alleviate CIPNP in-vivo, and therefore are provided additionally in combination with chemotherapeutics for the treatment of diseases such as cancer or other proliferative disorders. The CYP2J2 antagonists reduce chemotherapeutic induced pain and therefore allow for a higher dosing of the chemotherapeutic during cancer treatment. In addition the invention relates to the use of CYP2J2 agonists, or metabolites of CYP2J2, for sensitizing TRPV1. In this context the invention proposes to use combinations of CYP2J2 agonist or metabolites and transient receptor potential vanilloid 1 (TRPV1) agonists to treat disorders that respond to TRPV1 agonists, such as neuropathic pain.

FIELD OF THE INVENTION

The present invention pertains to novel treatments of neuropathic pain;in particular chemotherapy induced peripheral neuropathic pain (CIPNP).The invention provides antagonists cytochrome P450 epoxygenases (CYP),and more specifically antagonists of CYP2J2, as therapeutics for use inthe treatment of neuropathic pain such as CIPNP. CYP2J2 antagonists wereidentified to alleviate CIPNP in-vivo, and therefore are providedadditionally in combination with chemotherapeutics for the treatment ofdiseases such as cancer or other proliferative disorders. The CYP2J2antagonists reduce chemotherapeutic induced pain and therefore allow fora higher and better dosing of the chemotherapeutic during cancertreatment. In addition the invention relates to the use of CYP2J2agonists, or metabolites of CYP2J2, for sensitizing TRPV1. In thiscontext the invention proposes to use combinations of CYP2J2 agonist ormetabolites and transient receptor potential vanilloid 1 (TRPV1)agonists to treat disorders that respond to TRPV1 agonists, such asneuropathic pain.

DESCRIPTION

Neuropathic pain is a persistent or chronic pain syndrome that canresult from damage to the nervous system, the peripheral nerves, thedorsal root ganglion, dorsal root, or to the central nervous system.Neuropathic pain syndromes include allodynia, various neuralgias such aspost herpetic neuralgia and trigeminal neuralgia, phantom pain, andcomplex regional pain syndromes, such as reflex sympathetic dystrophyand causalgia. Causalgia is often characterized by spontaneous burningpain combined with hyperalgesia and allodynia. Tragically there is noexisting method for adequately, predictably and specifically treatingestablished neuropathic pain as present treatment methods forneuropathic pain consists of merely trying to help the patient copethrough psychological or occupational therapy, rather than by reducingor eliminating the pain experienced. Treatment of neuropathic or chronicpain is a challenge for physicians and patients since there are nomedications that specifically target the condition, and since themedications presently used result in only little relief and are based ontheir efficacy in acute pain conditions or on their efficacy onrelieving secondary effects like anxiety and depression. Incidence ofchronic pain is increasing in society and its burden on society is hugein both health care and lost productivity. Currently there are noscientifically validated therapies for relieving chronic pain. As aresult, the health community targets ‘pain management’ where multi-modaltherapies are used concurrently with the hope of providing someimprovement in quality of life. Thus, there is an urgent need for drugsthat can relieve chronic pain.

Chemotherapy induced peripheral neuropathic pain (CIPNP) is a severedose limiting side effect of cytostatics, such as taxanes, platinumderivates, vinca alkaloids and others. The symptoms usually start withtingling and can lead to burning, stabbing and aching pain as well ascold and mechanical allodynia. Due to CIPNP some patients stopanticancer therapy with cytostatics too early, resulting in a higherrisk of tumor progression. Unfortunately many promising substances, thatare already approved for the treatment of different kinds of neuropathicpain, such as gabapentin oramitriptyline seem to have little or noanalgesic effect in monotherapy of CIPNP. Understanding the cellular andmolecular mechanisms is necessary to treat or even prevent CIPNP and mayimprove the general success rate of cytostatic therapy.

Recent studies identified members of the transient receptorpotential-family of ion channels (TRPV1, TRPA1 and TRPV4) ascontributors to both mechanical and cold allodynia during oxaliplatinand paclitaxel-induced neuropathy. Activation or sensitization of TRPV1and TRPA1 can lead to enhanced release of CGRP and substance P both ofwhich can cause neurogenic inflammation and recruitment of T-cells.

However, it remains unclear which endogenous mediators are involved incytostatic-dependent activation or sensitization of TRP-channels, asneither of the cytostatics can directly activate TRP-channels.Interestingly, both paclitaxel and oxaliplatin are inducers ofCYP-epogenases (paclitaxel: CYP2C8, CYP2C9, oxaliplatin: CYP2E1,CYP1B1). Cytochrome P450 (CYP)-epoxygenases can metabolize ω-6 fattyacids, such as arachidonic acid (AA) and linoleic acid (LA) generatingeither lipid epoxides such as EETs (epoxyeicosatrienoid acids) orw-hydroxides such as 20-HETE.

The metabolism of arachidonic acid by cytochrome P450 monoxygenasesleads to the formation of various biologically active eicosanoids. Threetypes of oxidative reactions are known to occur. First, olefinepoxidation (catalyzed by epoxygenases) gives rise to theepoxyeicosatrienoic acids (EETs). Four important EET regioisomers are[5,6]-EET, [8,9]-EET, [11,12]-EET, and [14,15]-EET. The EETs arehydrolyzed by epoxide hydrolases to form the correspondingdihydroxyeicosatrienoic acids (DHETs). Second, omega terminal oxidationleads to the formation of omega terminal hydroxyeicosatetraenoic acids(HETEs). Third, allylic oxidation leads to the formation of midchainHETEs.

Several cytochrome P450 epoxygenases have been identified, includingmembers of the CYP1A, CYP2B, CYP2C, CYP2E, and CYP2J subfamilies.Attention has recently been focused on proteins of the CYP2J subfamily.One particular isoform, CYP2J2, is highly expressed in human cardiacmyocytes, where arachidonic acid is metabolized to produce EETs. CYP2J2proteins are also found in epithelial cells in the airway and in thegut. In contrast to the other P450 enzymes, CYP2J2 proteins aredistributed uniformly along the length of the gut, in epithelial andnon-epithelial cells. High levels of the CYP2J2 proteins are found incells of the autonomic ganglia, epithelial cells, and intestinal smoothmuscle cells. Several CYP2J homologues have been identified in animalsincluding rat CYP2J3, rat CYP2J4, mouse CYP2J5 and mouse CYP2J6.

Capsaicin is a highly selective agonist for transient receptor potentialvanilloid 1 receptor (TRPV1; formerly known as vanilloid receptor 1(VR1)), a ligand-gated, non-selective cation channel preferentiallyexpressed on small-diameter sensory neurons, especially those C-fiberswhich specialize in the detection of painful or noxious sensations.TRPV1 responds to noxious stimuli including capsaicin, heat, andextracellular acidification, and will integrate simultaneous exposuresto these stimuli. The initial effect of the activation ofTRPV1-expressing (capsaicin-sensitive) nociceptors are burningsensations, hyperalgesia, allodynia, and erythema. However, afterprolonged exposure to low-concentration capsaicin or single exposures tohigh-concentration capsaicin or other TRPV1 agonist, the small-diametersensory axons become less sensitive to a variety of stimuli, includingcapsaicin or thermal stimuli. This prolonged exposure is alsocharacterized by reduced pain responses. These later-stage effects ofcapsaicin are frequently referred to as “desensitization” and are therationale for the development of local capsaicin formulations for thetreatment of various pain syndromes and other conditions.

Therefore capsaicin, capsaicinoids and TRPV1 agonists may be useful foramelioration of a plurality of diseases. For example, they may be usedto treat neuropathic pain (including pain associated with diabeticneuropathy, postherpetic neuralgia, HIV/AIDS, traumatic injury, complexregional pain syndrome, trigeminal neuralgia, erythromelalgia andphantom pain), pain produced by mixed nociceptive and/or neuropathicmixed etiologies (e.g., cancer), osteoarthritis, fibromyalgia, lowerback pain, inflammatory hyperalgesia, vulvar vestibulitis or vulvodynia,sinus polyps interstitial cystitis, neurogenic or overactive bladder,prostatic hyperplasia, rhinitis, surgery, trauma, rectalhypersensitivity, burning mouth syndrome, oral mucositis, herpes (orother viral infections), prostatic hypertrophy, dermatitis, pruritis,itch, tinnitus, psoriasis, warts, cancers (especially skin cancers),headaches, and wrinkles.

Hence, until this day there is no specific therapy for neuropathic painavailable, in particular chemotherapy-induced peripheral neuropathicpain (CIPNP), which restricts the maximal dosing of chemotherapeuticagents during cancer treatment and causes severe impairment oflife-quality of patients undergoing chemotherapy. The object of thepresent invention is therefore to provide a novel treatment option totackle neuropathic pain, specifically CIPNP.

The above problem is solved in a first aspect by a cytochrome P450epoxygenase (CYP)-antagonist for use in the prevention or treatment ofpain in a subject. In some embodiments of the invention theCYP-antagonist is selected from the group consisting of a CYP1A-, CYP2B,CYP2C-, CYP2E-, and preferably a CYP2J-antagonist. Most preferably theCYP-antagonist is an antagonist of a mammalian homologue of CYP2J2(CYP2J2-antagonist), preferably human CYP2J2, such as telmisartan,aripiprazole or most preferably terfenadine.

Encompassed by the present invention is the use of any CYP2J2antagonist, preferably a selective CYP2J2 antagonist. The term“selective CYP2J2 antagonist” pertains to antagonists of CYP2J2 thatselectively inhibit activity, function or expression of CYP2J2 but notof other related enzymes such as for example CYP3A molecules. In orderto identify whether a candidate antagonist is a CYP2J2 antagonist, aluminogenic cytochrome P450 glow assay can be employed. CYP proteinscatalyse the formation of arachidonic acid metabolites. Luminogenic CYPassays use prosubstrates for the light-generating reaction ofluciferase. CYPs convert the prosubstrates to luciferin or a luciferinester, which produces light in a second reaction with a luciferasereaction mix called Luciferin Detection Reagent (LDR). The amount oflight produced in the second reaction is proportional to CYP activity.

In order to test selectivity of a candidate CY2J2 antagonist,luminogenic CYP assays specific for other CYP enzymes such as CYP3A4 canbe employed. Comparing the inhibitory activity of a candidate antagonistagainst CYP2J2 with the inhibitory activity of the same antagonistagainst another CYP protein such as CYP3A4, therefore providesinformation about the selectivity of the candidate antagonist.

Preferred selective CYP2J2 antagonists in context of the presentinvention are selected from the group of the herein newly disclosedCYP2J2 antagonists consisting of estradiol, phenoxybenzamine-HCl,loratadine, clobetasol propionate, doxazosin mesylate, fenofibrate,levonorgestrel, aripiprazole, halcinonide, telmisartan, clofazimine,levothyroxine-Na, alosetron-HCl, fluocinonide, liothyronine-Na,meclizine dihydrochloride and terfenadine and derivatives thereof.

In context of the herein described invention said pain to be treated ispreferably neuropathic pain (including pain associated with diabeticneuropathy, postherpetic neuralgia, HIV/AIDS induced neuropathic pain,traumatic injury, complex regional pain syndrome, trigeminal neuralgia,erythromelalgia and phantom pain), pain produced by mixed nociceptiveand/or neuropathic mixed etiologies (e.g., cancer), osteoarthritis,fibromyalgia, lower back pain, inflammatory hyperalgesia, vulvarvestibulitis or vulvodynia, sinus polyps interstitial cystitis,neurogenic or overactive bladder, prostatic hyperplasia, rhinitis,surgery, trauma, rectal hypersensitivity, burning mouth syndrome, oralmucositis, herpes (or other viral infections), prostatic hypertrophy,dermatitis, pruritis, itch, tinnitus, psoriasis, warts, cancers,headaches, and wrinkles, central pain due to stroke or mass lesion,spinal cord injury, or multiple sclerosis. However, most preferredembodiments pertain to chemotherapy-induced peripheral neuropathic pain(CIPNP).

The present invention now provides a pain therapy comprising theinhibition of the activity of in particular CYP2J2 which produces themetabolic compound 9,10-EpOME—according to the invention, a sensitizerof ion channel-mediated pain perception. Surprisingly, the inhibition ofCYP2J2 in accordance with the invention proved to be effective in-vivoto alleviate neuropathic pain induced by paclitaxel in a mouse model,indicating the use of CYP2J2 antagonists as analgesic againstneuropathic pain, in particular CIPNP.

One further embodiment of the invention relates to the above mentionedprevention or treatment of pain, which comprises the administration ofsaid CYP antagonist of the invention to a subject suffering from saidpain, and wherein said subject received, receives or will receivechemotherapy. Therefore, the subject is in preferred embodiments asubject suffering from, or diagnosed with, a cancer disease.

Chemotherapy in context of the invention preferably involves theadministration of a chemotherapeutic agent to a subject in need of sucha treatment selected from pyrimidinone-based anti-neoplastic agents suchas cytarabine, 5-flurouracil or platin agents, such as cisplatin, ortaxanes, such as paclitaxel, docetaxel or cabazitaxel, or derivativesthereof. Such chemotherapeutic agents are known to induce neuropathicpain, in particular this is known for taxanes, which are thereforepreferred in context of the invention. Most preferred is paclitaxel.

Further, said prevention or treatment of pain in accordance with theinvention comprises the concomitant or sequential administration of saidCYP antagonist and said chemotherapeutic agent. See for this embodimentalso the description below for a combination of the invention.

The problem of the invention is solved in another aspect by a9,10-epoxy-12Z-octadecenoic acid (9,10-EpOME)-antagonist for use in theprevention or treatment of pain in a subject. 9,10-EpOME was found to begenerated by CYP activity. Therefore, instead of antagonizing CYP, theinventive result may be alternatively achieved by antagonizing the9,10-EpOME directly in order to avoid a sensitization of pain mediatingneurons. Such 9,10-EpOME-antagonists of the invention are preferablysmall molecules but also proteins or peptides (e.g. antibodies orfragments thereof) binding to 9,10-EpOME and inhibiting thesensitization of TRPV1.

For this aspects the above described specific embodiments for the use ofCYP antagonists apply also for 9,10-EpOME-antagonists of the invention,in particular the embodiments relating to said prevention or treatmentand the chemotherapy.

The problem is additionally solved by a combination comprising (i) a CYPantagonist or an 9,10-EpOME-antagonist and (ii) a chemotherapeutic agentfor concomitant or sequential use in the prevention or treatment of adisease, wherein the disease is selected from a proliferative disorder,such as cancer, or pain, such as CIPNP.

The term “proliferative disorder” is used herein in a broad sense toinclude any disorder that requires control of the cell cycle, forexample cardiovascular disorders such as restenosis and cardiomyopathy,auto-immune disorders such as glomerulonephritis and rheumatoidarthritis, dermatological disorders such as psoriasis,anti-inflammatory, antifungal, antiparasitic disorders such as malaria,emphysema and alopecia. In these disorders, the compounds of the presentinvention may induce apoptosis or maintain stasis within the desiredcells as required. Preferably, the proliferative disorder is a cancer orleukaemia, most preferably cancer of the breast, lung, prostate,bladder, head and neck, colon, ovarian cancer, uterine cancer, sarcomaor lymphoma.

The present embodiment also relates to the treatment of a subject groupsuffering from pain, wherein the subjects are under the treatment with achemotherapeutic. The CYP-antagonist of the invention therefore may beadministered during the same period of time as the cancer treatment, oralternatively is done before or after, which can be preferable in orderto avoid accumulating adverse effects. The inventive result is achievedwhen the physiological effects of a CYP-antagonist of the invention andthe pain inducement of a chemotherapeutic are combined in a subject inneed of such a treatment. After the last dose of a medicament isadministered during therapy, the physiological effects induced by themedicament will not diminish immediately, but most likely later.Therefore, using the antagonists of the invention in sequentialtherapeutic cycles, for example the antagonists of the invention areadministered in advance of a chemotherapy instead at the same time,still leads to a combination of the clinical effects of both compoundsin the patient, and therefore falls under the meaning of the combinationtherapy of the present invention.

The term “combination” means in this context an active substancecombination of two or more active substances in a formulation and alsoas a combination in the sense of individual formulations of the activesubstances administered at specified intervals from one another in atherapeutic treatment. Thus the term “combination” shall include theclinical reality of a co-administration of two or more therapeuticallyeffective compounds, as it is described in context of the presentinvention.

Co-administration: In the context of the present application,co-administration of two or more compounds is defined as administrationof the two or more compounds to the patient within one year, includingseparate administration of two or more medicaments each containing oneof the compounds as well as simultaneous administration whether or notthe two or more compounds are combined in one formulation or whetherthey are in two or more separate formulations.

The combination of the invention in one embodiment includes that (i) and(ii) are combined by sequential or concomitant administration to asubject during said prevention or treatment, preferably wherein theantagonists and chemotherapeutics are concomitantly administered duringsaid prevention or treatment.

A chemotherapeutic is preferably selected from pyrimidinone-basedanti-neoplastic agents such as cytarabine, 5-flurouracil or platinagents, such as cisplatin, or taxanes, such as paclitaxel or docetaxel,or derivatives thereof. Most preferably the chemotherapeutic ispaclitaxel or docetaxel.

Antagonists of the herein described invention are preferably selectedfrom the group of compounds consisting of inhibitory RNA, inhibitoryantibodies or fragments thereof, and/or small molecules. Herein below adetailed description of preferred CYP-antagonists is provided.

In context of the invention it is also preferred that at least oneadditional therapeutic effective against pain, for example a morphine,an opioid or a non-opioid analgesic or other analgesic, is administeredto said subject.

In another aspect of the invention there is provided a method for theprevention or treatment of pain in a subject, the method comprising thestep of administering to said subject a therapeutically effective amountof a CYP-antagonist or a 9,10-EpOME-antagonist in accordance with thepresent invention. The CYP-antagonist is preferably selected from thegroup consisting of a CYP1A-, CYP2B-, CYP2C-, CYP2E-, andCYP2J-antagonist. The CYP2J-antagonist is preferably an antagonist of amammalian homologue of CYP2J2 (CYP2J2-antagonist), preferably, humanCYP2J2, such as terfenadine or telmisartan, as well as biosimilars orderivatives thereof.

Other preferred CYP antagonists are selected from the group consistingof estradiol, phenoxybenzamine-HCl, loratadine, clobetasol propionate,doxazosin mesylate, fenofibrate, levonorgestrel, aripiprazole,halcinonide, telmisartan, clofazimine, levothyroxine-Na, alosetron-HCl,fluocinonide, liothyronine-Na, meclizine dihydrochloride andterfenadine.

The diseases treatable in context of the afore-described methods aredescribed herein above.

During the treatment or prevention it is preferred that at least oneadditional therapeutic effective against pain is administered to saidpatient, such as other analgesics, for example an opioid or a non-opioidanalgesic.

An additional aspect of the invention then relates to a method forincreasing sensitivity of Transient Receptor Potential Vanilloid 1(TRPV1) in a subject, comprising administering to said subject atherapeutically effective amount of 9,10-EpOME or of an CYP2J2 agonist.

In context of the invention is was surprisingly found that 9,10-EpOMEsensitises the TRPV1 channel protein, which is a major mediator of painperception. Therefore, the present invention in a preferred embodimentprovides 9,10-EpOME as an TRPV1 agonist, which is particularly useful inmedicine. Combinations of 9,10-EpOME and the TRPV1 agonist capsaicinsignificantly enhanced capsaicin activity. One embodiment for examplepertains to the treatment of a disease characterized by a pathologicalsuppressed sensation of pain or insensitivity of pain.

In another embodiment 9,10-EpOME may be used in a method for enhancingthe activity of TRPV1-agonists, such as capsaicin. Capsaicin is used asan analgesic in topical ointments, nasal sprays, and dermal patches torelieve pain. It may be applied in cream form for the temporary reliefof minor aches and pains of muscles and joints associated witharthritis, backache, strains and sprains, often in compounds with otherrubefacients. It is also used to reduce the symptoms of peripheralneuropathy such as post-herpetic neuralgia caused by shingles.

The mechanism by which capsaicin's analgesic and/or anti-inflammatoryeffects occurs is purportedly by mimicking a burning sensation;overwhelming the nerves by the calcium influx, leading todesensitisation and/or apoptosis of nociceptors and thereby renderingthe nerves unable to report pain for an extended period of time. Withchronic exposure to capsaicin, nociceptors of neurons underwentapoptosis, leading to reduction in sensation of pain and blockade ofneurogenic inflammation. If capsaicin is removed, the nociceptiveneurons recover over time. Therefore, the use of 9,10-EpOME of theinvention may greatly increase the medical effects of capsaicin andrelated compounds, or alternatively may help to reduce capsaicin dosing.

Therefore, in a preferred embodiment of the invention there is provideda method for treating a disease in a subject, comprising theadministration of a therapeutically effective amount of (i) 9,10-EpOMEor of an CYP2J2 agonists, and (ii) an TRPV1 agonist. With regard tosequential or concomitant uses of therapeutics, reference is made to theabove descriptions which equally apply for this aspect of the invention.

A disease is preferably selected from neuropathic pain (including painassociated with diabetic neuropathy, postherpetic neuralgia, HIV/AIDS,traumatic injury, complex regional pain syndrome, trigeminal neuralgia,erythromelalgia and phantom pain), pain produced by mixed nociceptiveand/or neuropathic mixed etiologies (e.g., cancer), osteoarthritis,fibromyalgia, lower back pain, inflammatory hyperalgesia, vulvarvestibulitis or vulvodynia, sinus polyps interstitial cystitis,neurogenic or overactive bladder, prostatic hyperplasia, rhinitis,surgery, trauma, rectal hypersensitivity, burning mouth syndrome, oralmucositis, herpes (or other viral infections), prostatic hypertrophy,dermatitis, pruritis, itch, tinnitus, psoriasis, warts, cancers,headaches, and wrinkles. Generally any disease is comprised which istreatable by an TRPV1 agonist.

Exemplary and preferred TRPV1 agonist of the invention are selected fromthe group consisting of capsaicin, piperine, 6-gingerol, 6-shogaol,α-sanshool, β-sanshool, γ-sanshool, δ-sanshool, hydroxyl α-sanshool, andhydroxyl β-sanshool.

Another aspect then pertains to a 9,10-EpOME or an CYP2J2 agonists foruse in a method as described herein above.

Yet another aspect pertains to a combination of (i) 9,10-EpOME or of anCYP2J2 agonist, and (ii) an TRPV1 agonist, for use in medicine,preferably in the treatment of a disease selected from neuropathic pain(including pain associated with diabetic neuropathy, postherpeticneuralgia, HIV/AIDS, traumatic injury, complex regional pain syndrome,trigeminal neuralgia, erythromelalgia and phantom pain), pain producedby mixed nociceptive and/or neuropathic mixed etiologies (e.g., cancer),osteoarthritis, fibromyalgia, lower back pain, inflammatoryhyperalgesia, vulvar vestibulitis or vulvodynia, sinus polypsinterstitial cystitis, neurogenic or overactive bladder, prostatichyperplasia, rhinitis, surgery, trauma, rectal hypersensitivity, burningmouth syndrome, oral mucositis, herpes (or other viral infections),prostatic hypertrophy, dermatitis, pruritis, itch, tinnitus, psoriasis,warts, cancers (especially skin cancers), headaches, and wrinkles.

The TRPV1 agonist is preferably selected from the group consisting ofcapsaicin, piperine, 6-gingerol, 6-shogaol, α-sanshool, β-sanshool,γ-sanshool, δ-sanshool, hydroxyl α-sanshool, and hydroxyl β-sanshool.

A subject in accordance with the herein described invention ispreferably a mammal, preferably a human, most preferably a humanreceiving a chemotherapeutic treatment, such as a cancer patient.

CYP-Antagonists

“CYP antagonists” in context of the present invention are preferablyselected from the group consisting of a CYP1A-, CYP2B-, CYP2C-, CYP2E-,and more preferably a CYP2J-antagonist. Most preferably theCYP-antagonist is an antagonist of a mammalian homologue of CYP2J2(CYP2J2-antagonist), preferably human CYP2J2. Therefore, in mostpreferred embodiments and aspects of the herein described invention theterm “CYP-antagonist” is a CYP2J2 antagonists, or antagonists ofmammalian homologs of human CYP2J2.

As used herein, the term “CYP-antagonist” means a substance that affectsa decrease in the amount or rate of CYP expression or activity. Such asubstance can act directly, for example, by binding to CYP anddecreasing the amount or rate of CYP expression or activity. ACYP-antagonist can also decrease the amount or rate of CYP expression oractivity, for example, by binding to CYP in such a way as to reduce orprevent interaction of CYP with a CYP receptor; by binding to CYP andmodifying it, such as by removal or addition of a moiety; and by bindingto CYP and reducing its stability. A CYP-antagonist can also actindirectly, for example, by binding to a regulatory molecule or generegion so as to modulate regulatory protein or gene region function andaffect a decrease in the amount or rate of CYP expression or activity.Thus, a CYP-antagonist can act by any mechanisms that result in decreasein the amount or rate of CYP expression or activity.

A CYP-antagonist can be, for example, a naturally or non-naturallyoccurring macromolecule, such as a polypeptide, peptide, peptidomimetic,nucleic acid, carbohydrate or lipid. A CYP-antagonist further can be anantibody, or antigen-binding fragment thereof, such as a monoclonalantibody, humanized or human antibody, chimeric antibody, minibody,bifunctional antibody, single chain antibody (scFv), variable regionfragment (Fv or Fd), Fab or F(ab)2. A CYP-antagonist can also bepolyclonal antibodies specific for CYP. A CYP-antagonist further can bea partially or completely synthetic derivative, analog or mimetic of anaturally occurring macromolecule, or a small organic or inorganicmolecule.

A CYP-antagonist that is an antibody can be, for example, an antibodythat binds to CYP and inhibits binding to a CYP receptor, or alters theactivity of a molecule that regulates CYP expression or activity, suchthat the amount or rate of CYP expression or activity is decreased. Anantibody useful in a method of the invention can be a naturallyoccurring antibody, including a monoclonal or polyclonal antibodies orfragment thereof, or a non-naturally occurring antibody, including butnot limited to a single chain antibody, chimeric antibody, bifunctionalantibody, complementarity determining region-grafted (CDR-grafted)antibody and humanized antibody or an antigen-binding fragment thereof.

A CYP-antagonist that is a nucleic acid can be, for example, ananti-sense nucleotide sequence, an RNA molecule, or an aptamer sequence.An anti-sense nucleotide sequence can bind to a nucleotide sequencewithin a cell and modulate the level of expression of CYP, or modulateexpression of another gene that controls the expression or activity ofCYP. Similarly, an RNA molecule, such as a catalytic ribozyme, can bindto and alter the expression of the CYP gene, or other gene that controlsthe expression or activity of CYP. An aptamer is a nucleic acid sequencethat has a three dimensional structure capable of binding to a moleculartarget.

A CYP-antagonist that is a nucleic acid also can be a double-strandedRNA molecule for use in RNA interference methods. RNA interference(RNAi) is a process of sequence-specific gene silencing bypost-transcriptional RNA degradation, which is initiated bydouble-stranded RNA (dsRNA) homologous in sequence to the silenced gene.A suitable double-stranded RNA (dsRNA) for RNAi contains sense andantisense strands of about 21 contiguous nucleotides corresponding tothe gene to be targeted that form 19 RNA base pairs, leaving overhangsof two nucleotides at each 3′ end (Elbashir et al., Nature 411:494-498(2001); Bass, Nature 411:428-429 (2001); Zamore, Nat. Struct. Biol.8:746-750 (2001)). dsRNAs of about 25-30 nucleotides have also been usedsuccessfully for RNAi (Karabinos et al., Proc. Natl. Acad. Sci. USA98:7863-7868 (2001). dsRNA can be synthesized in vitro and introducedinto a cell by methods known in the art.

Preferred CYP2J2 antagonists are selected from the group consisting ofestradiol, phenoxybenzamine-HCl, loratadine, clobetasol propionate,doxazosin mesylate, fenofibrate, levonorgestrel, aripiprazole,halcinonide, telmisartan, clofazimine, levothyroxine-Na, alosetron-HCl,fluocinonide, liothyronine-Na, meclizine dihydrochloride andterfenadine.

Compositions and Kits for Treating or Preventing Pain or OtherNeurological Disorders

Another aspect of the present application relates to compositions andkits for treating or preventing pain or proliferative disorder by usingthe compounds or combinations of the invention. In one embodiment, thecomposition comprises compounds as described herein above, wherein thecompounds are preferably selected from an antibody, antibody fragment,short interfering RNA (siRNA), aptamer, synbody, binding agent, peptide,aptamer-siRNA chimera, single stranded antisense oligonucleotide,triplex forming oligonucleotide, ribozyme, external guide sequence,agent-encoding expression vector, small molecule and a pharmaceuticallyacceptable carrier.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary agents can also be incorporated into the compositions. Incertain embodiments, the pharmaceutically acceptable carrier comprisesserum albumin.

The pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intrathecal, intra-arterial,intravenous, intradermal, subcutaneous, oral, transdermal (topical) andtransmucosal administration.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine; propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a neuregulin) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forsustained or controlled release of the active ingredient. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures:

FIG. 1: Concentrations of oxidized linoleic acid metabolites duringpaclitaxel CIPNP or inflammation. Shown are the concentrations of9,10-EpOME (a) and 12,13-EpOME (b) in sciatic nerves, DRG and the spinaldorsal horn 24 h after i.p. injection of vehicle (black) or paclitaxel(grey, 6 mg-kg-1) in C57B16/N mice; n.d.: not determined. Concentrationsof 9-HODE (c) and 13-HODE (d) in sciatic nerves, L4-L6-DRGs and thecorresponding section of the spinal dorsal horn 24 h after i.p.injection of vehicle (black) or paclitaxel (grey) in C57B16/N mice. (e)Relation of 9,10-EpOME-concentrations in L4-L6-DRGs and thecorresponding section of the dorsal horn 24 h after intraplar injectionof zymosan (12.5 mg/ml, 20 μl) Data represent the mean±SEM of 8-10animals per group; ***p<0.001, student's t-test.

FIG. 2: Direct effects of 9,10-EpOME on DRG-neurons. (a) Application of9,10-EpOME [10 μM, 30 s] causes calcium transients on DRG neurons whichrespond to high potassium (50 mM KCl, 30 s). A representative trace isshown. (b) Dose response relationship of 9,10-EpOME dependent calciumincreases in DRG neurons related to the number of responding neuronsData represents the mean±SEM of five measurements per concentration. (c)and (d) Calcium transients caused by 9,10-EpOME [10 μM, 30 s] can bedisrupted using calcium-free medium containing EGTA (2 mM) washed in 2minutes before and after 9,10-EpOME stimulation. Data represents themean±SEM of 24 (calcium-free) or 16 (control) neurons. (e) and (f)Calcium transients of 9,10-EpOME [10 μM, 30 s] can be blocked by aselective TRPV1 antagonist (AMG 9810, 1 μM) but not by a selective TRPA1antagonist (HC-030031, 20 μM) washed in for two minutes prior to thesecond 9,10-EpOME stimulation. Data represents the mean±SEM of 16(control), 31 (AMG 9810) or 18 (HC-030031) neurons; **p<0.01, student'st-test.

FIG. 3: 9,10-EpOME dose-dependently sensitizes TRPV1 in DRG neurons andpotentiates capsaicin-induced increases in spontaneous EPSC frequency inlamina II neurons of spinal cord slices. (a) DRG neurons weredouble-stimulated with capsaicin (200 nM, 15 s each) and incubated witheither vehicle or 9,10-EpOME [1 μM] for two minutes prior to the secondcapsaicin stimulation. (b) Dose-dependent difference in ratio betweenthe first and the second capsaicin response using the same protocol asdescribed in (a). Data represent the mean±SEM of the following number ofneurons: 27 (control), 26 (250 nM 9,10-EpOME), 21 (500 nM 9,10-EpOME),19 (750 nM 9,10-EpOME), 41 (1 μM 9,10-EpOME), 18 (2 μM 9,10-EpOME) or 28(using 50 μM AITC for 20 s instead of capsaicin); *p<0.05,**p<0.01,***p<0.001 student's t-test. (c) Traces of spontaneous EPSCs(sEPSCs) in lamina II neurons. Low panel, traces 1, 2, 3, and 4 areenlarged and indicate recordings of baseline, 1st capsaicin (1 mM),9,10-EpOME (1 mM), and 2nd capsaicin (1 mM) plus 9,10-EpOME,respectively. (d) Frequency of sEPSCs. Compared to baseline of sEPSCs,capsaicin induced profound increases in sEPSC frequency (from 6.9±0.4 Hzand 13.7±0.4 Hz). Treatment of 9,10-EpOME alone slightly increased thefrequency of sEPSCs (8.2±0.8 Hz) and significantly potentiated the sEPSCfrequency increase by capsaicin (18.7±1.1 Hz). *P<0.05, compared with notreatment baseline; #P<0.05, compared with 1st capsaicin treatment (1mM). n=5 neurons/group. (E) Amplitude of sEPSCs. Capsaicin and9,10-EpOME had no significant effects on sEPSC amplitude. n=5neurons/group.

FIG. 4: TRPV1-sensitization by 9,10-EpOME in DRG neurons is mediated bya Gs-coupled receptor and the cAMP-PKA pathway. (a) 9,10-EpOME catalyzedthe [γ-35S]-GTP binding in membrane fractions of rat DRGs. Experimentswere carried out using membrane fractions of rat DRGs in the presence of30 μM GDP and vehicle (Methyl Acetate. 0.7% (v/v)), adenosine [10 μM] or9,10-EpOME [1 μM] for 30 minutes. The data were obtained from 3measurements of membrane fractions from a total of 15 animals. DRGs fromfive animals were pooled for each measurement; *p<0.05, **p<0.01,Kruskal-Wallis test with Dunn's multiple comparison post hoc test. (b)Concentrations of cAMP in neuron-enriched DRG cultures after stimulationwith 9,10-EpOME, cicaprost or forskolin (1 μM each) for 15 minutes. Datarepresents mean±SEM of DRG cultures from from five mice. (c) and (d)TRPV1 sensitization by 9,10-EpOME [1 μM] can be reduced by preincubationwith a PKA-inhibitor (H89-dihydrochloride, 10 μM for 1 h). Datarepresent mean±SEM of 15 (vehicle), 19 (EpOME) or 33 neurons (EpOME withH89 preincubation). (e) and (f) TRPV1 sensitization by 9,10-EpOME [1 μM]is not affected by preincubation with a PKC-inhibitor (GF 109203X, 10 μMfor 1 h). Data represent mean±SEM of 18 (vehicle), 23 (EpOME) or 39neurons (EpOME with GFX preincubation); *p<0.05, **p<0.01 student'st-test; n.s. not significant.

FIG. 5: Intraplantar or intrathecal injection of 9,10-EpOME reduces painthresholds and sensitizes capsaicin induced mechanical thresholds inwild type mice. (a) and (b) C57B1/6N mice received an intraplantarinjection of 9,10-EpOME (10 μM) or vehicle (DMSO 0.3% (v/v) in saline).Thermal (a) or mechanical (b) thresholds were monitored for 5 h postinjection. Data represents mean±SEM from eight mice. (c) and (d) Wildtype BL/6N mice were injected intrathecally with 9,10-EpOME (10 μM) orvehicle (DMSO 0.3% (v/v) in saline). Thermal (c) or mechanical (d)thresholds were monitored for 2 h (thermal) or 3 h (mechanical) postinjection with 15 minute intervals for the first hour and 30 minuteintervals for the second hour. Data represents mean±SEM from eight mice.Die Abbildungen sind nicht mitgeliefert

FIG. 6: Release of iCGRP from isolated sciatic nerves or neuron enrichedDRG cultures after 9,10-EpOME stimulation. (a) Release of iCGRP fromisolated sciatic nerves of wild type BL/6N mice, stimulated with thefollowing solutions for 5 minutes each: synthetic intestinal fluid(SIF), SIF+EpOME (1 μM) or vehicle (DMSO 0.03% (v/v)), SIF+EpOME (orvehicle)+capsaicin (500 nM), SIF. Data represents mean±SEM from sixindividual sciatic nerves. (b) Release of iCGRP from neuron enriched DRGcultures after stimulation with either PBS, 9,10-EpOME, capsaicin or9,10-EpOME+capsaicin for 15 minutes; a: 9,10-EpOME 1 μM, b: capsaicin400 nM, c: 9,10-EpOME 2.5 μM. Data represents mean±SEM of DRG culturesfrom six mice; #,*p<0.05, **p<0.01, ***p<0.001 student's t-test. Dashedline indicates assay sensitivity.

FIG. 7: CYP2J6 is upregulated during paclitaxel-induced neuropathicpain. (a) Time-course of the mechanical thresholds of wild typeC57B^(1/6)N-mice after injection of paclitaxel (6 mg·kg-1 i.p.). bl:baseline, data represents mean±SEM of ten mice per group. After eightdays sciatic nerves, DRGs and the spinal dorsal horn were dissected. (b)Expression of murine CYP-epoxygenase-transcripts eight days afterpaclitaxel-injection (6 mg·kg-1 i.p.). Data represents mean±SEM from theDRGs of four mice per group, *p<0.05, **p<0.01, student's t-test. (c)Concentrations of 9,10-EpOME in sciatic nerves, DRG and the spinaldorsal horn eight days after i.p. injection of vehicle (black) orpaclitaxel (grey, 6 mg·kg-1) in C57B16/N mice; **p<0.01, student'st-test. (d) Scheme of eicosanoid- and linoleic acid metabolite-synthesisin murine DRGs eight to nine days after paclitaxel-treatment as revealedby LC-MS/MS analysis. Structures were obtained from lipidmaps.org.

FIG. 8: Inhibition of CYP2J6 by terfenadine reduces lipid concentrationsand ameliorates paclitaxel-induced CIPNP in vivo. (a) Levels of9,10-EpOME shown in % of control determined by LC-MS/MS in sciaticnerves, DRGs and the dorsal horn of the spinal cord eight days aftertreatment with paclitaxel (6 mg·kg-1 i.p. and 1 mg·kg-1 terfenadine(grey) or vehicle (2% DMSO v/v, black)) Data represents mean±SEM fromthe DRGs of five mice per group; *p<0.05, **p<0.01, student's t-test.(b) Remaining concentrations of all measured epoxylipids anddihydro-metabolites (9,10-EpOME, 12,13-EpOME, 9,10-DiHOME, 12,13-DiHOMEand 14,15-EET) in sciatic nerve, DRGs, dorsal horn of the spinal cordand plasma after administration of terfenadine (1 mg·kg-1). (c)Mechanical thresholds of mice treated with paclitaxel for eight days (6mg·kg-1 i.p.) that received an intravenous injection of terfenadine (1or 2 mg kg-1) or vehicle (DMSO 2.5 or 5% (v/v)). The mechanicalthresholds were monitored up to 5 h after injection of terfenadine orvehicle. Data represent mean±SEM from 8-9 mice per group; #,*p<0.05,two-way ANOVA with Bonferroni post hoc test (*1 mg kg-1, #2 mg kg-1terfenadine). (d) Mechanical thresholds of mice eight days afterpaclitaxel-injection (6 mg·kg-1 i.p.) that received an intravenousinjection of Loratadine (1 mg kg-1) or vehicle (DMSO (2.5% (v/v)). Datarepresent mean±SEM from 6-9 mice per group.

FIG. 9: Correlation of calculated inhibition values of CYP2J2 andCYP3A4. Antagonists located in the upper left quadrant are selective forCYP2J2. Luminogenic CYP2J2 assays were conducted according to themanufacturer's protocol(https://www.promega.de/resources/pubhub/enotes/cytochrome-p450-2j2-enzyme-assay-using-a-novel-bioluminescent-probe-substrate/).In order to test selectivity of a candidate CY2J2 antagonist, additionalluminogenic CYP assays specific for CYP3A4 were employed and theinhibitory activity of a candidate CYP2J2 antagonists was compared tothe inhibitory activity of the same antagonist against CYP3A4.

SEQ ID NO: 1 TO 14: PRIMER SEQUENCES EXAMPLES Materials and MethodsAnimals

All animal experiments were performed according to the recommendationsin the Guide for the Care and Use of Laboratory Animals of the NationalInstitutes of Health and approved by the local Ethics Committees forAnimal Research (Darmstadt) with the permit number F95/42. For allbehavioral experiments the inventor's used only 6-12 weeks old maleC57BL/6N mice purchased from commercial breeding companies (CharlesRiver, Sulzfeld, Germany, Janvier, Le Geneset-Saint-Isle, FR). Tocompare mechanical thresholds the inventor's used age and sex matchedlittermates as control.

Prostanoid-receptor deficient mice (DP1−/−, IP−/−, EP2−/− and EP4−/−)were bred in the Institute of Clinical Pharmocology, Frankfurt, asdescribed previously.

Paclitaxel Model of Chemotherapy-Induced Neuropathic Pain

Paclitaxel was solved in Cremophor EL/Ethanol 1:1 and diluted in saline.The dose for intraperitoneal injection was set to 6 mg/kg as describedpreviously.

Behavioral Tests

For the determination of mechanical allodynia or thermalhypersensitivity, mice were kept in test cages on an elevated grid forat least 2 h to allow accommodation. Baseline measurements wereperformed using a Dynamic Plantar Aesthesiometer or a HargreavesApparatus (Ugo Basile, Comerio, VA, Italy) detecting withdrawal latencyof the hind paws after mechanical stimulation. For the assessment of themechanical thresholds, the steel rod was pushed against the mid-plantarhind paw with linear ascending force (0-5 g over 10 seconds, increasing0.5 g/s) until a fast withdrawal response occurred. Slow movements ofthe paw were not counted. Paw withdrawal latencies (PWL) were determinedin seconds (s)±0.1 with a cut-off time of 20 s. The non-injected andinjected paws were measured alternately in intervals of 5-10 min. Fordetermination of thermal thresholds, mice were kept in test cages on awarmed glass plate (32° C.) for at least 2 h on the first day to allowaccommodation. Then, the mid-plantar region of the paws was stimulatedwith a radiant heat device, consisting of a high intensity projectorlamp, until withdrawal occurred. The non-injected and injected paws weremeasured alternately in intervals of 5-10 min. For all behavioral teststhe investigator was blinded for treatment or genotype of the mice.

Treatments: For peripheral injections, 20 μl of 9,10-EpOME [5 μM](Cayman, Ann Arbor, Mich., USA) were injected subcutaneously (s.c.) inthe mid-plantar area of the hind paw. Control animals received thecorresponding volumes of DMSO (Sigma, Deisenhofen, Germany; 1.6% (v/v)in Saline). For intrathecal injections, 5 μl of 9,10-EpOME [10 μM] in3.2% DMSO/saline (v/v) were injected by direct lumbar puncture in awake,conscious mice as described previously. Terfenadine or Loratadine (bothfrom Tocris, Bristol, UK) were injected intravenously in the tail vein.

Primary Dorsal Root Ganglia (DRG) Cultures

Murine DRGs were dissected from spinal segments and directly transferredto ice cold HBSS with CaCl2 and MgCl2 (Invitrogen, Carsbad, Calif.,USA). Next, isolated DRGs were incubated with collagenase/dispase (500U/ml Collagenase; 2.5 U/ml Dispase) in neurobasal medium containingL-glutamine [2 mM] penicillin (100 U/ml), streptomycin (100 μg/ml), B-27and gentamicin (50 μg/ml) (all from Invitrogen, Carlsbad, Calif., USA)at 37° C. for 75 min. After removal of the collagenase/dispase-solution,cells were washed twice with neurobasal medium containing 10% FCS andincubated for 10 min with 0.05% trypsin (Invitrogen, Carlsbad, Calif.,USA). The washing steps were repeated and the cells were mechanicallydissociated with a 1 ml Gilson pipette. Finally, the neurons were platedon poly-1-lysine (Sigma, Deisenhofen, Germany) coated glass cover slipsand incubated with neurobasal medium containing L-glutamine [2 mM]penicillin (100 U/ml), streptomycin (100 μg/ml), B-27 and gentamicin (50μg/ml) over night until assessment by calcium imaging.

Calcium Imaging Experiments

Calcium-Imaging experiments were performed with two different setups.First, the inventor's used an Axioscope 2 upright microscope (Zeiss,Jena, Germany) with a 10× Achroplan water immersion objective (Zeiss).The microscope was equipped with an Imago CCD camera and a Polychrome IVmonochromator (all TILL Photonics, Gräfelfing, Germany). Images wereacquired every 2 seconds at both wavelengths (340 nm and 380 nm) andprocessed using the Tillvision software 23. Later, a LeicaCalcium-imaging setup was used, consisting of a Leica DMI 4000 binverted microscope equipped with a DFC360 FX (CCD-) camera, Fura-2filters and an N-Plan 10×/0.25 Ph1 objective (all from LeicaMicrosystems, Wetzlar, Germany). Images were taken every 2 seconds andprocessed with the LAS AF-software. For each experiment the inventor'schose an area with large cell numbers and monitored 40-110 cellssimultaneously. Calcium-Imaging experiments were performed usingDRG-neurons 24-48 hours after preparation. Cells were loaded with 5 μMfura-2-AM-ester and 0.02% Pluronic F127 (both Biotium, Hayward, Calif.and incubated for 30 to 60 min. at 37° C. Then, the cells were washedwith external solution (containing in mM: NaCl [145], CaCl2 [1.25],MgCl2 [1], KCl [5], D-glucose [10], HEPES [10]; adjusted to pH 7.3).Baseline measurements were performed in external solution at a flow rateof 1-2 ml/min. Calcium free solutions were generated by removal of CaCl2and addition of EGTA [2 mM] and osmotically controlled by increasingNaCl concentrations to 150 mM. Stock solutions of HC-030031 (Sigma,Deisenhofen, Germany), AMG 9810, H89-dihydrochloride, 8-bromo-cAMP, GF109203X (all from Tocris, Bristol, UK) and NGF (Merck Millipore,Darmstadt, GE) were diluted in external solution to their finalconcentrations.

Quantitative Real-Time PCR

Lumbal DRGs were dissected from mice at indicated time points and RNAwas extracted using the mirVana™ miRNA Isolation Kit (Ambion, lifetechnologies, Carlsbad, Calif., USA). Reverse transcription andReal-time PCR were prefomed using the TaqMan® system (life technologies,Carlsbad, Calif., USA) and evaluated with the ΔΔC(T)-method as describedpreviously 24,25. The following oligonucleotides were used foramplification of cDNA:

TABLE 1Primer sequences used for quantitative real-time PCR from murine tissue, a =MGH primer bank, ID: 160948617c2. Gene Forward Reverse CYP2C295′GCCTCAAAGCCTACTGTCA-3′ (SEQ ID NO 1) 5′-AACGCCAAAACCTTTAATC-3′(SEQ ID NO 2) CYP2C37 5′-ATACTCTATATTTGGGCAGG-3′ (SEQ ID NO 3)5′-GTTCCTCCACAAGGCAAC-3′ (SEQ ID NO 4) CYP2C38 5′-TTGCCTTCTGTAATCCCCC-3′(SEQ ID NO 5) 5′-TCTAACGCAGGAATGGATAAAC-3′ (SEQ ID NO 6) CYP2C395′-GGAGACAGAGCTGTGGC-3′ (SEQ ID NO 7) 5′-TAAAAACAATGCCAAGGCCG-3′(SEQ ID NO 8) CYP2C44 5′-CTTTCCAACGAGCGATTCCC-3′ (SEQ ID NO 9)5′-TGTTTCTCCTCCTCGATCTTGC-3′ (SEQ ID NO 10) CYP2J65′-GGCCTCCCACCTAGTGGAA-3′ (SEQ ID NO 11) 5′-ATAACCTCGTCCAGTAACCTCA-3′(SEQ ID NO 12) CYP3A11 5′-GACAAACAAGCAGGGATGGAC-3′ (SEQ ID NO5′-CCAAGCTGATTGCTAGGAGCA-3′ (SEQ ID NO 14) 13)

Determination of EETs by Liquid Chromatography-Tandem Mass Spectrometry(LC-MS/MS)

Sample extraction and standards: Sample extraction was performed asdescribed previously. Briefly, stock solutions with 2500 ng/ml of allanalytes were prepared in methanol. Working standards were obtained byfurther dilution with a concentration range of 0.1-250 ng/ml for EETs,EpOMEs and DiHOMEs and HODEs Sample extraction was performed withliquid-liquid-extraction. Therefore tissue or cell culture medium wasextracted twice with 600 μl ethyl acetate. The combined organic phaseswere removed at a temperature of 45° C. under a gentle stream ofnitrogen. The residues were reconstituted with 50 μl ofmethanol/water/(50:50, v/v), centrifuged for 2 min at 10,000×g and thentransferred to glass vials (Macherey-Nagel, Duren, Germany) prior toinjection into the LC-MS/MS system.

Instrumentation for measuring epoxylipids and HODEs: The LC-MS/MS systemconsisted of an API 4000 triple quadrupole mass spectrometer (AppliedBiosystems, Darmstadt, Germany), equipped with a Turbo-V-sourceoperating in negative ESI mode, an Agilent 1100 binary HPLC pump anddegasser (Agilent, Waldbronn, Germany) and an HTC Pal autosampler(Chromtech, Idstein, Germany) fitted with a 25 μL LEAP syringe (AxelSemrau GmbH, Sprockhövel, Germany). High purity nitrogen for the massspectrometer was produced by a NGM 22-LC-MS nitrogen generator (cmcInstruments, Eschborn, Germany). For the chromatographic separation aGemini NX C18 column and precolumn were used (150 mm×2 mm i. d., 5 μmparticle size and 110 Å pore size from Phenomenex, Aschaffenburg,Germany). A linear gradient was employed at a flow rate of 0.5 ml/minmobile phase with a total run time of 17.5 minutes. Mobile phase A waswater/ammonia (100:0.05, v/v) and B acetonitrile/ammonia (100:0.05,v/v). The gradient started from 85% A to 10% within 12 min. This washeld for 1 min at 10% A. Within 0.5 min the mobile phase shifted back to85% A and was held for 3.5 min to equilibrate the column for the nextsample. The injection volume of samples was 20 Quantification wasperformed with Analyst Software V 1.4.2 (Applied Biosystems, Darmstadt,Germany) employing the internal standard method (isotope-dilution massspectrometry). Ratios of analyte peak area and internal standard area(y-axis) were plotted against concentration (x-axis) and calibrationcurves were calculated by least square regression with 1/concentration 2weighting.

[35S] GTPγS Binding Assays

To measure activation of a putative of a G-protein coupled receptor,GTPγS binding assays were performed with membrane preparations of DRGsfrom adults rats using 1 μM 9,10-EpOME (Cayman, Ann Arbor, Mich., USA)and fresh [35S] GTPγS (1250 Ci/mmol, Perkin Elmer, Waltham, Mass., USA).

Measurement of iCGRP

CGRP-measurements were performed as described previously 32 using aCGRP-enzyme immune assay kit (SpiBio, Bertin pharma, France). ForCGRP-measurements from DRG cultures, DRGs of wild type BL/6N mice weredissected and treated as described above and cultured overnight in 48well plates.

Data Analysis and Statistics

All data are presented as mean±s.e.m. To determine statisticallysignificant differences in all behavioral experiments analysis ofvariance (ANOVA) for repeated measures was used followed by post hocBonferroni correction using GraphPad Prism. For in vitro experimentscomparing only two groups, student's t-test was carried out. P<0.05 wasconsidered as statistically significant.

Cytochrome P450 Luciferase Assays

CYP2J2 and CYP3A4 Glo assays were performed according to manufacturersinstructions (P450-Glo™, Promega).

Protocol of the CYP2J2 Assay:

-   -   Preparation of the CYP2J2-enzyme (2 nM)/Luciferin-2J2/4F12        substrate (2 μM) mix 5 μl/well using MultiDrop,    -   Addition 50 nl/well of compounds (10 μM end concentration)/DMSO        (0.5% end concentration) using Echo    -   Incubation for 30 min at 37° C.    -   Addition of NADPH regeneration solution 5 μl/well using        MultiDrop    -   Incubation for 30 min at 37° C.    -   Addition of LDR-esterase solution 10 μl/well using MultiDrop    -   Incubation for 30 min at 37° C., Luminescence readout on        EnSpire.

Protocol of the CYP3A4-Assay:

-   -   Preparation of the CYP3A4-enzyme (2 nM)/Luciferin-IPA substrate        (7 μM) mix 5 μl/well using MultiDrop,    -   Addition 50 μl/well of compounds (10 μM end concentration)/DMSO        (0.5% end concentration) using Echo,    -   Incubation for 30 min at 37° C.,    -   Addition of NADPH regeneration solution 5 μl/well using        MultiDrop,    -   Incubation for 30 min at 37° C.,    -   Addition of LDR-esterase solution 10 μl/well using MultiDrop,    -   Incubation for 30 min at 37° C. and Luminescence readout on        EnSpire.

Example 1: CYP-Derived Lipids in Chemotherapy Induced Neuropathic Pain

To investigate, whether or not CYP-derived lipids may play a role inchemotherapy-induced neuropathic pain, the inventors injected paclitaxelor vehicle in wild type BL/6N mice and dissected the sciatic nerves,DRGs and the spinal dorsal horn 24 h post injection. Lipidconcentrations were determined using LC-MS/MS. It was found that theconcentrations of the oxidized linoleic acid metabolite 9,10-EpOME (FIG.1A) but not of its sister lipid 12,13-EpOME (FIG. 1B) or theirdihydro-metabolites 9,10- and 12,13-DiHOME (see Supplementary FIG. 1) isstrongly elevated in DRGs respectively (FIG. 1A). Also quantified wasthe levels of 9- and 13-HODE (FIG. 1C, 1D), which are generated duringinflammatory pain and are endogenous activators of TRPV1 33. However,the inventors could not detect any difference in their levels followingpaclitaxel treatment. To investigate whether the increased9,10-EpOME-concentration in DRGs is specific for paclitaxel treatment,zymosan was injected in the hind paw of wild type BL/6N mice in order toinduce inflammatory pain. The L4-L6-DRGs and the corresponding sectionof the dorsal horn were dissected 24 h post injection at the peak ofinflammation. Lipid quantification by LC-MS/MS did not reveal anydifference in 9,10-EpOME levels during inflammatory pain (FIG. 1E).

Next, the inventors characterized 9,10-EpOME concerning its effects onDRG-neurons in calcium imaging experiments. The inventors observed, thata short stimulation of 30 s with 10 μM 9,10-EpOME caused a calciumtransient in DRG neurons (FIG. 2A). The inventors performed doseresponse analysis to investigate the potency of 9,10-EpOME in evokingcalcium transients and found a maximum of 10.3% of DRG neuronsresponding to 25 μM of 9,10-EpOME with no significant increase in thepercentage of responding neurons to higher concentrations (FIG. 2B). Toanalyze, whether the 9,10-EpOME evoked calcium transients result fromrelease of intracellular calcium stores of from influx of extracellularcalcium, the inventors used calcium-free external solution, containing 2mM EGTA and stimulated DRG neurons twice with 10 μM 9,10-EpOME for 30 s.Two minutes before the second stimulation, calcium-free externalsolution was washed in and the neurons did not respond to 9,10-EpOME anymore, thus indicating influx of external calcium caused by 9,10-EpOME(FIGS. 2C, 2D). The positive control for neurons was a final stimulationwith 50 mM KCl for 30 s.

To identify the involved ion channel, selective antagonists of TRPV1(AMG 9810, 1 μM) and TRPA1 (HC-030031, 20 μM) were used in order toblock the calcium flux caused by 9,10-EpOME. DRG neurons were stimulatedtwice with 9,10-EpOME (10 μM, 30 s) and the cells were pre-incubatedwith the TRP channel antagonists for two minutes prior to the second9,10-EpOME stimulus. The inventors observed, that the selective TRPV1antagonist AMG 9810, but not the TRPA1 antagonist HC-030031 could blockthe second 9,10-EpOME-evoked calcium transient, indicating TRPV1 astargeted channel by 9,10-EpOME (FIGS. 2E, 2F).

Example 2: 9,10-EpOME Sensitizes TRPV1

Next, the inventors analyzed if 9,10-EpOME was also capable ofsensitizing TRPV1 or TRPA1 in a lower and more physiologicalconcentration (1 μM). The inventors therefore stimulated DRG neuronstwice with capsaicin (200 nM, 15 s) and incubated the cells for twominutes with 9,10-EpOME [1 μM] or vehicle prior to the second capsaicinstimulus and observed a significantly stronger response of DRG neuronsto capsaicin that were incubated with 9,10-EpOME, thus indicatingsensitization of TRPV1 by 9,10-EpOME (FIG. 3A). To investigate thepotency of 9,10-EpOME dependent TRPV1 sensitization, dose responseanalysis was performed using 9,10-EpOME concentrations from 250 nM to 2μM. It was observed that a dose dependent increase in the amplitudes ofthe second capsaicin responses compared to vehicle. This effect seems tobe specific for TRPV1, because mustard oil-dependent TRPA1-responsescould not be sensitized by 9,10-EPOME [1 μM] (FIG. 2B).

To confirm the effect of TRPV1 sensitization by 9,10-EpOME withelectrophysiological means, the inventors measured sEPSCs from lamina IIneurons of spinal cord slices using two capsaicin stimulations [1 μM]and incubating the cells prior to the second capsaicin stimulus with9,10-EpOME [1 μM] (FIG. 3C). Treatment of 9,10-EpOME alone slightlyincreased the frequency of sEPSCs. In combination with capsaicin,however, the sEPSC frequency was strongly potentiated (FIG. 3D).However, no difference in the amplitude of sEPSCs could be observed witheither 9,10-EpOME, TRPV1 or the combination of both substances (FIG.3E).

Since it is known that lipid mediated TRPV1-sensitization mostlyinvolves activation of a G-protein coupled receptor, the inventor'sperformed GTPγS assays to analyze whether 9,10-EpOME is capable ofactivating a GPCR in DRGs and observed a significantly increased signalof GTPγS after incubation with 1 μM 9,10-EpOME (FIG. 4A). To identifythe mechanism of 9,10-EpOME mediated TRPV1 sensitization, the inventor'snext measured cAMP in neuron enriched DRG cultures that were stimulatedwith either vehicle, 9,10-EpOME, the IP-receptor agonist cicaprost orforskolin [1 μM each] for 15 minutes. Interestingly, the inventor'sobserved that 9,10-EpOME caused a significant increase in cAMPconcentrations compared to its vehicle (FIG. 4B). These results indicateactivation of a Galphas coupled receptor by 9,10-EpOME.

Since TRPV1 can be phosphorylated by PKA and PKC, both resulting inincreased activity and sensitization of the channel 35, the inventorsinvestigated whether inhibitors of PKA or PKC can reduce9,10-EpOME-evoked TRPV1-sensitization in calcium-imaging experimentswith cultured DRG neurons from wild type BL/6N mice. The inventors couldreproduce the capsaicin-dependent TRPV1 sensitization using the sameprotocol as mentioned above with double capsaicin stimulation and anin-between incubation with 9,10-EpOME. However, the inventors observedthat pre-incubation with a PKA-inhibitor (H89 dihydrochloride, 10 μM for1 h) caused a significant reduction of 9,10-EpOME-evoked TRPV1sensitization. (FIGS. 4C, 4D). The use of the PKC-inhibitor GF 109203X(GFX) under the same conditions (10 μM, preincubation for 1 h) did nothave any effect on 9,10-EpOME derived TRPV1 sensitization (FIGS. 4E,4F), thus pointing toward PKA- but not PKC-mediated TRPV1 sensitizationby 9,10-EPOME.

The inventors then tested the Galphas-coupled prostanoid receptors fortheir potential involvement in 9,10-EpOME dependent TRPV1 sensitizationin calcium imaging experiments. Prostanoid receptors have varyingspecificity for their ligand prostanoids and may as well be activated byother lipids. However, the inventors could not observe any reduction in9,10-EpOME evoked TRPV1-sensitization in the DRGs of eitherProstaglandin E receptors EP2 and EP4 or prostaglandin D- or I-receptor(DP- and IP-receptor) deficient mice (not shown). To characterize invivo effects of 9,10-EpOME, the inventors injected the lipid in hindpaws of wild type BL/6N mice and measured the thermal (FIG. 5A) andmechanical thresholds (FIG. 5B) up to 5 h post injection. In both cases,9,10-EpOME caused a significant reduction of the pain thresholds lasting1 h (thermal) or 2 h (mechanical) after injection (FIG. 1A, 1B). Theinventors then injected 9,10-EpOME intrathecally and measured thermaland mechanical thresholds in short time intervals. A significant butrather weak reduction of the thermal thresholds 30 minutes after i.th.injection was observed (FIG. 5C). However, the mechanical thresholdswere decreased for up to 1.5 h after i.th. injection of 9,10-EpOME (FIG.5D).

Since increased activity of TRPV1 causes increased release of calcitoningene related peptide (CGRP) promoting neurogenic inflammation 37, theinventors analyzed whether 9,10-EpOME is capable of increasing TRPV1dependent CGRP release. The inventors dissected sciatic nerves from wildtype BL/6N mice and incubated them with 9,10-EpOME alone [1 μM], ortogether with capsaicin [500 nM] and observed a strong increase of CGRPrelease with co-stimulation of capsaicin and 9,10-EpOME. TheCGRP-release was significantly greater than using only capsaicin or9,10-EpOME (FIG. 6A). To investigate if this effect is also visible inthe cell somata, neuron enriched DRG-cultures with either 9,10-EpOME,capsaicin, or both substances were stimulated using two differentEpOME-concentrations [1 and 2.5 μM]. Again, the release of iCGRP wassignificantly increased using both EpOME and capsaicin than with eitherof the substances. However, there was no significant increase inCGRP-release using 2.5 μM of 9,10-EPOME (FIG. 6B).

Example 3: CYP2J2 Regulates 9,10-EpOME

Next it was investigated how 9,10-EpOME synthesis is regulated duringpaclitaxel CIPNP. Since 9,10-EpOME is supposed to be synthesized byCYP-epoxygenases of the subfamilies 2C and 2J 16,38, the inventorsexamined the expression of murine CYP-expoxygenases of thesesubfamilies. Eight days after paclitaxel treatment, the inventorsobserved a stable plateau in the mechanical thresholds of paclitaxeltreated mice (FIG. 7A).

The inventors then dissected DRGs of vehicle and paclitaxel treated miceand investigated the expression of murine CYP2C29, CYP2C37, CYP2C38,CYP2C39, CYP2C44, CYP2J6 and CYP3A11. However, CYP isoforms 2C29 and2C44 could not be detected in murine DRGs. The inventors observed thatCYP2J6 showed the strongest expression in the DRGs of paclitaxel treatedmice compared to vehicle treatment (FIG. 7B). This increased expressionin CYP2J6 correlates with increased levels of 9,10-EpOME eight daysafter paclitaxel treatment, as analyzed by LC-MS/MS measurement ofsciatic nerve, lumbar DRGs and the spinal cord.

Example 4: CYP2J2 Antagonists Inhibit 9,10-EpOME Synthesis and ReduceCIPNP

Terfenadine, a potent inhibitor of the human CYP2J2, which is theanalogue protein of murine CYP2J6, was used as an antagonist. Since theinteraction sites of Terfenadone and the human CYP2J2 have already beendescribed, the inventors aligned the amino acids of the murine CYP2J6and the human CYP2J2 and found all putative interaction sites withTerfenadone (Leu83, Met116, Ile127, Phe30, Thr315, Ile376, Leu378,Va1380, Leu402 and Thr488) at the same position in both proteins exceptArg117 which is exchanged to glutamine. Based on the surprising aminoacid sequence similarity between CYP2J2 and CYP2J6 Terfenadine interactsas well with CYP2J6 and inhibits the protein. To investigate the effectsof Terfenadine on lipid levels, the inventors injected mice that hadreceived paclitaxel eight days before with 1 mg·kg-1 Terfenadine i.v.After two hours, the inventors dissected the sciatic nerve, DRGs and thedorsal spinal cord and quantified epoxylipids in these tissues. Theinventors could observe a significant reduction of the 9,10-EpOMEconcentrations in all investigated tissues (FIG. 8A). The inventors alsoobserved, that the remaining concentrations of all measured epoxylipidsand their (9,10-EpOME, 12,13-EpOME, 9,10-DiHOME, 12,13-DiHOME and14,15-EET) were reduced significantly in DRGs, the spinal dorsal hornand the plasma, but not in the sciatic nerve of Terfenadine treatedanimals respectively (FIG. 8B).

The inventors next investigated whether treatment with Terfenadine mayreduce Paclitaxel-induced CIPNP in mice. Therefore, the inventor'sinjected Terfenadine (1 or 2 mg·kg-1 or vehicle (DMSO) intravenously inmice that had already received paclitaxel eight days before. Theinventors measured the mechanical thresholds of mice 1, 2, 4 and 5 hpost Terfenadine injection and could observe a significant increase inmechanical thresholds of mice that were treated with Terfenadine,lasting for 2 h. However, no significant differences between the twodoses could be observed (FIG. 8C). Since Terfenadine is ahistamine-1-receptor antagonist, the inventors used Loratadine, anotherH1-receptor-antagonist that does not inhibit CYP2J2, to investigate, ifthe antinociceptive effects are really caused by inhibition of CYP2J2,or the histamine-1-receptor. However, treatment with Loratadine did notreduce paclitaxel-induced CIPNP compared to the vehicle (FIG. 8D).

Example 5: Screening of New Selective CYP2J2-Antagonists

The Screens-Well® FDA Approved Drug Library v2 was screened for newselective antagonists of CYP2J2 for use in the context of the hereindescribed invention. The enzymatic CYP-Glo luciferase based reaction wasused to assay the activity of CYP2J2 and as unselective control CYP3A4.Tefenadine was used as positive control in the experiments. The Resultsfrom both screens are depicted in FIG. 9. Antagonists that showed over60% inhibition against CYP2J2 and about 0% inhibition of CYP3A4 areregarded as selective CYP2J2 antagonists and are useful for the methodsand uses as described herein, and are listed in table 2 below:

TABLE 2 Average Average Compound inhibition inhibition ID CYP2J2 (%)CYP3A4 (%) Name c054 84.4 12.1 Estradiol c089 75.0 17.9Phenoxybenzamine•HCl c124 80.8 −23.5 Loratadine c146 78.2 −4.3Clobetasol Propionate c244 73.7 −43.3 Doxazosin Mesylate c246 78.0 −26.2Fenofibrate c314 64.0 5.1 Levonorgestrel c337 92.8 15.5 Aripiprazolec440 76.8 −18.5 Halcinonide c485 89.5 7.6 Telmisartan c516 79.1 −82.9Clofazimine c542 87.6 12.9 Levothyroxine•Na c595 81.8 14.0 Alosetron•HClc596 75.9 10.7 Fluocinonide c606 93.6 −6.8 Liothyronine•Na c608 71.517.4 Meclizine Dihydrochloride

Discussion

9,10-EpOME is capable of sensitizing TRPV1 in DRG neurons via a cAMP-PKAdependent mechanism in submicromolar concentrations, leading tosubsequent release of iCGRP from DRGs. Other oxidized linoleic acidmetabolites (OLAMs), such as 9 and 13-HODE, which are produced duringexcessive heating of skin, have already been shown to be direct TRPV1agonists and to contribute to inflammatory hyperalgesia. The inventorscould also detect 9- and 13-HODE in murine tissue, most predominantly inperipheral tissues.

The inventors used the CYP2J2-inhibitor Terfenadine to reduce synthesisof 9,10-EpOME and could reduce the levels of epoxylipids to about 50%.Treatment with Terfenadine resulted in reduced mechanicalhypersensitivity in mice during paclitaxel CIPNP. Antagonists of CYP2J2and its homologs are therefore useful for treating or preventing CIPNP,which was confirmed because animals that were treated with Loratadine, aselective H1-receptor antagonist, that does not affect CYP2J2, did notshow an improvement in paclitaxel CIPNP, thus indicating that the effectthat was observed with Terfenadine is due to inhibition of CYP2J2 andnot of the histamine 1-receptor.

Chemotherapy-induced neuropathic pain and subsequent sensorydysfunctions still remain the most severe side effects of cytostatics.Especially during paclitaxel-treatment, an early acute pain syndrome canbe observed which seems to be mediated by sensitization of nociceptiveneurons. However, there is no information available on endogenousmediators that may contribute to this pathophysiological state.According to the inventor's data, 9,10-EpOME-dependent TRPV1sensitization and increased activity of nociceptive neurons may thuscontribute to paclitaxel acute pain syndrome (P-APS).

Currently, there is a strong unmet medical need for CIPNP therapeutics.Treatment of patients with antioxidants or neuroprotextive substances,such as amifostine or glutathione failed to ameliorate CIPNP in largerandomized and placebo controlled clinical trials, and a recent Cochranereview concludes, that there is currently no evidence for functionalCIPNP therapy with these substances. Moreover, antioxidants mayinterfere with the antineoplastic effects of cytostatics. Recently, itwas reported that treatment with N-acetyl cysteine (NAC) and vitamin Eincreased lung tumor cell proliferation and tumor growth in mice byreducing DNA damage. In this regard, CYP2J2-inhibitors may be superiorover using antioxidants, because they have been reported to even reducecancer growth in vitro and in vivo by activating caspase-3, Bax andBcl-2 and by reducing tumor cell migration and adherence.

1. A cytochrome P450 epoxygenase (CYP)-antagonist for use in the prevention or treatment of neuropathic pain in a subject, wherein said CYP antagonist is a CYP2J-antagonist.
 2. The CYP antagonist for use according to claim 1, wherein said neuropathic pain is selected from the group consisting of post-herpetic neuralgia, trigeminal neuralgia, focal peripheral nerve injury, and anesthesia dolorosa, central pain due to stroke or mass lesion, spinal cord injury, or multiple sclerosis, and peripheral neuropathy due to diabetes, HIV, or chemotherapy.
 3. The CYP antagonist for use according to claim 1, wherein said pain is chemotherapy-induced peripheral neuropathic pain (CIPNP).
 4. The CYP antagonist for use according to claim 1, wherein said CYP antagonist is a CYP2J2 antagonist selected from the group consisting of estradiol, phenoxybenzamine-HCl, loratadine, clobetasol propionate, doxazosin mesylate, fenofibrate, levonorgestrel, aripiprazole, halcinonide, telmisartan, clofazimine, levothyroxine-Na, alosetron-HCl, fluocinonide, liothyronine-Na, meclizine dihydrochloride and terfenadine.
 5. A 9,10-epoxy-12Z-octadecenoic acid (9,10-EpOME)-antagonist for use in the prevention or treatment of neuropathic pain in a subject.
 6. The 9,10-EpOME-antagonist according to claim 5, wherein said pain is chemotherapy-induced peripheral neuropathic pain (CIPNP).
 7. A combination comprising (i) a CYP antagonist or an 9,10-EpOME-antagonist and (ii) a chemotherapeutic agent for concomitant or sequential use in the prevention or treatment of a disease, wherein the disease is selected from a proliferative disorder, such as cancer, or pain, such as CIPNP.
 8. The combination for use according to claim 7, wherein (i) and (ii) are combined by sequential or concomitant administration to a subject during said prevention or treatment, preferably wherein the antagonists are concomitantly administered during said prevention or treatment.
 9. The CYP-antagonist for use according to claim 1, the 9,10-EpOME-antagonist for use in the prevention or treatment of neuropathic pain in a subject or the combination, comprising (i) a CYP antagonist or an 9,10-EpOME-antagonist and (ii) a chemotherapeutic agent for concomitant or sequential use in the prevention or treatment of a disease, wherein the disease is selected from a proliferative disorder, such as cancer, or pain, such as CIPNP, wherein said antagonists are selected from the group of compounds consisting of inhibitory RNA, inhibitory antibody, and/or small molecule.
 10. The CYP-antagonist for use according to claim 1, wherein at least one additional therapeutic effective against pain is administered to said subject.
 11. A 9,10-EpOME or a CYP2J-agonist, for use in the treatment of a disease in a subject.
 12. The 9,10-EpOME or the CYP2J-agonist for use according to claim 11, wherein said subject received, receives or will receive a therapy with a transient receptor potential vanilloid 1 (TRPV1)-agonist.
 13. A combination comprising (i) 9,10-EpOME or of an CYP2J-agonists, and (ii) an TRPV1-agonist, for use in medicine,
 14. The 9,10-EpOME or the CYP2J-agonist for use according to claim 11, or the combination comprising (i) 9,10-EpOME or of an CYP2J-agonists, and (ii) an TRPV1-agonist, wherein said disease is selected from neuropathic pain (including pain associated with diabetic neuropathy, postherpetic neuralgia, HIV/AIDS, traumatic injury, complex regional pain syndrome, trigeminal neuralgia, erythromelalgia and phantom pain), pain produced by mixed nociceptive and/or neuropathic mixed etiologies (e.g., cancer), osteoarthritis, fibromyalgia, lower back pain, inflammatory hyperalgesia, vulvar vestibulitis or vulvodynia, sinus polyps interstitial cystitis, neurogenic or overactive bladder, prostatic hyperplasia, rhinitis, surgery, trauma, rectal hypersensitivity, burning mouth syndrome, oral mucositis, herpes (or other viral infections), prostatic hypertrophy, dermatitis, pruritis, itch, tinnitus, psoriasis, warts, cancers (especially skin cancers), headaches, and wrinkles.
 15. The 9,10-EpOME or the CYP2J-agonist for use according to claim 11, or the combination comprising (i) 9,10-EpOME or of an CYP2J-agonists, and (ii) an TRPV1-agonist, wherein said TRPV1 agonist is selected from the group consisting of capsaicin, piperine, 6-gingerol, 6-shogaol, α-sanshool, β-sanshool, γ-sanshool, δ-sanshool, hydroxyl α-sanshool, and hydroxyl β-sanshool.
 16. The 9,10-EpOME-antagonist for use according to claim 5 wherein at least one additional therapeutic effective against pain is administered to said subject.
 17. The combination for use according to claim 7, wherein at least one additional therapeutic effective against pain is administered to said subject. 